Direct observation of dynein-microtubule binding

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In thee outer doublet microtubule in the flagellar axoneme, the outer dynein arms are arranged with a 24 nm interval, while each of the seven kinds of inner arms are arranged with a 96 nm interval. How such a regular arrangement is produced is almost entirely unknown. In this study, we aimed to approach this mechanism by directly observing the dynein-microtubule binding process. Using total reflection optics and fluorescently labeled proteins, we observed that the association of dynein to the microtubule is a cooperative process, quantified the cooperative binding using outer-dynein-arm-docking complex (ODA-DC) expressed in insect culture cells. Along with these studies, we searched for proteins responsible for the binding of inner dynein arms and discovered that the protein tektin is involved in the attachment of dynein e in Chlamydomonas axonemes. This is the first study to show tektin involvement in the dynein arrangement ; tektin, first discovered in sea urchin sperm flagella, has been thought to function as a ruler that determines the longitudinal periodicity in the axoneme. In a still different line of experiment, we examined the dynamic nature of dynein-microtubule association using a technique called fluorescence recovery after photobleaching. Using electroporation-mediated incorporation of fluorescently labeled dynein subunit, we found that inner arm dynein of Chlamydomonas is constantly being turned over within the flagella. These findings should provide important insights into the mechanism as to how dynein arms are attached to axonemal microtubules.